1. Field of the Invention
The present invention relates generally to methods of using genetically modified microorganisms for the manufacture of useful chemical compounds (metabolic engineering) and more specifically to constructing microbial strains by genetic manipulation that are capable of converting readily available carbon sources, such as D-glucose, into a more valuable product, for example, xylitol.
2. Related Art
Xylitol is a chemical compound of a considerable value as a special sweetener. It is approximately as sweet as sucrose, non-toxic, and non-cariogenic.
Currently, xylitol is produced by chemical hydrogenation of D-xylose. D-xylose is obtained from hydrolysates of various plant materials where it is always present in a mixture with other pentoses and hexoses. Purification of xylose and also xylitol presents therefore a significant problem. A number of processes of this type are known. U.S. Pat. Nos. 3,784,408, 4,066,711, 4,075,406, and 4,008,285 can be mentioned as examples.
The reduction of D-xylose into xylitol can also be achieved in a microbiological process using either strains isolated from nature (Barbosa, M. F. S. et al., J. Industrial Microbiol. 3: 241-251 (1988)) or genetically engineered strains (Hallborn, J. et al., Biotechnology 9: 1090-1095 (1991)). However, obtaining the substrate, D-xylose, in a form suitable for yeast fermentation is also a considerable problem because inexpensive xylose sources such as sulphite liquor from pulp and paper processes contain impurities which inhibit yeast growth.
An attractive alternative method for the manufacture of xylitol would be obtaining it by fermentation of a cheap and readily available substrate, such as D-glucose. However, no microorganisms are known that produce xylitol in significant amounts during one-step fermentation of any common carbon sources other than D-xylose and D-xylulose, both of which are structurally very closely related to xylitol.
On the other hand, many microorganisms, especially osmophilic yeasts, for example Zygosaccharomyces rouxii, Candida polymorpha, and Torulopsis candida, produce significant amounts of a closely related pentitol, D-arabitol, from D-glucose (Lewis D. H. & Smith D. C., New Phytol. 66: 143-184 (1967)). Using this property of osmophilic yeasts, H. Onishi and T. Suzuki developed a method for converting D-glucose into xylitol by three consecutive fermentations (Appl. Microbiol. 18: 1031-1035 (1969)). In this process, D-glucose was first converted into D-arabitol by fermentation with an osmophilic yeast strain. Second, the D-arabitol was oxidized into D-xylulose in a fermentation with Acetobacter suboxydans. Finally, the D-xylulose was reduced to xylitol in the third fermentation using one of many yeast strains capable of reducing D-xylulose into xylitol.
An obvious disadvantage of this method is that it involves three different fermentation steps, each taking from 2 to 5 days; further additional steps like sterilization and cell removal are also needed, thus increasing processing costs. The yield of the step fermentation process is low and the amount of by-products is high. Thus, a need still exists for methods for the economical production of xylitol in microbial systems from readily available substrates.